Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

Abstract

Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.

Fig. 1.

At low ionic strength (50 mM NaCl), topologically closed DNA overstretches via formation of melting bubbles. (A) Schematic depiction of topologically closed λ-DNA (Upper), along with the corresponding force-distance curves [Lower, recorded with a pulling/relaxing speed of ∼3 μm/s for extension and retraction of the relative DNA length (L/L_c)]. Gray spheres represent optically trapped beads; orange spheres depict biotin-streptavidin linkages between the DNA molecule and the trapped beads. (B) Schematic illustration (Upper) of the same construct shown in A, but with nicks in the DNA backbone. The corresponding force-distance curves are also shown (Lower, pulling/relaxing speed ∼3 μm/s). (C) Corresponding fluorescence images of EGFP-RPA and Sytox for the construct shown in A. (D) Analogous fluorescence images to those displayed in C, but obtained with a nicked DNA construct similar to that illustrated in B. Strand unpeeling is highlighted by dashed orange arrows. Force-distance curves in A and B were recorded in the absence of fluorescent probes.

Fig. 2.

At low ionic strength (50 mM NaCl), melting bubbles nucleate in AT-rich regions. (A) Selection of fluorescence images of EGFP-RPA binding to topologically closed λ-DNA molecules as a function of relative DNA extension (L/L_c). A different DNA molecule was used for each extension. (B) Composite fluorescence images displaying the binding of EGFP-RPA (in green) and Sytox (in red). DNA molecules shown here are the same as those in A. (C) Variation in fluorescence intensity of EGFP-RPA along the DNA molecule (in green) for L/L_c = 1.04 and 1.53, respectively. Black traces display the AT-content of λ-DNA (in 100-bp bins) and are oriented in the direction that best matches the RPA fluorescence intensity (SI Text, Correlating Melting-Bubble Formation with AT-Rich DNA). These, and 10 further examples, are also shown in Fig. S5.

Fig. 3.

Observable melting bubbles disappear with increasing salt concentration. (A) Selection of composite fluorescence images from sequential binding of EGFP-RPA (green) and Sytox (red) to topologically closed λ-DNA molecules as a function of salt concentration. In each case, a separate DNA molecule was extended midway into its overstretching plateau (L/L_c = 1.35) and exposed to (i) 50 mM NaCl, (ii) 150 mM NaCl, (iii) 150 mM NaCl plus 2 mM MgCl₂, and (iv) 150 mM NaCl plus 20 mM MgCl₂. (B) Fractional coverage of RPA (revealing melting bubbles), Sytox (labeling B-DNA), and unstained DNA for the four images reported in A. Each data point has an associated error of ≤10% (SI Text, Estimating the Fraction of DNA Bound by Fluorescent Probes). Note that the salt concentration used in the far left image of A is the same as that used in Figs. 1 and 2.

Fig. 4.

Increasing ionic strength suppresses strand unpeeling in topologically open DNA during overstretching. (A) Force-extension measurements of DNA molecules with identical sequence at different monovalent salt concentrations, ranging from 50 mM to 1 M NaCl. Inset: Schematic of the DNA construct used, containing only one unconstrained DNA end. Gray spheres represent optically trapped beads; orange spheres represent biotin-streptavidin linkages between the DNA molecule and the trapped beads. (B) Divalent salt titration of the force-extension behavior of DNA molecules with the same sequence as in Fig. 4A. Monovalent salt concentration: 150 mM NaCl. The divalent salt was increased from 2 mM to 20 mM MgCl₂. All data in this figure were obtained using a 8,350-kb DNA fragment (14). Compared with experiments on λ-DNA, the presence of a single melting front, the more heterogeneous sequence, and the slower stretching speed used (10 nm/s) allow for identification of stick-slip dynamics that are not resolved in Fig. 1B (recorded with λ-DNA).

Fig. 5.

At high ionic strength (150 mM NaCl plus 20 mM MgCl₂), S-DNA formation is favored over strand unpeeling in topologically open DNA. (A) Selection of composite fluorescence images from sequential binding of EGFP-RPA (green) and Sytox (red) to λ-DNA (with one free end on each side of the molecule) as a function of relative DNA extension (L/L_c). Data obtained at 150 mM NaCl plus 20 mM MgCl₂. The white circles indicate regions of RPA binding. (B) Fractional coverage of RPA, Sytox, and unstained regions on the DNA molecule for each end-to-end distance reported in A. Each data point has an associated error of ≤10% (SI Text, Estimating the Fraction of DNA Bound by Fluorescent Probes). (C) Comparison of fluorescence images from sequential binding of EGFP-RPA and Sytox recorded under low-salt conditions (50 mM NaCl), illustrating domains of both unpeeling and melting bubbles in λ-DNA with a single nick in the phosphate backbone. Images obtained under mild buffer flow (∼1 μm/s); the orange circle denotes the unpeeled tract of ssDNA bound by RPA.